Alternative Antigen Processing and Presentation Pathways by Tumors - 03

8/9/2019 Alternative Antigen Processing and Presentation Pathways by Tumors - 03

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The handle http://hdl.handle.net/1887/22802 holds various files of this Leiden Universitydissertation

Author: Cunha Oliveira, Claudia daTitle: Alternative antigen processing and presentation pathways by tumorsIssue Date: 2013-12-10

http://hdl.handle.net/1887/22802http://hdl.handle.net/1887/22802http://hdl.handle.net/1887/22802http://hdl.handle.net/1887/22802https://openaccess.leidenuniv.nl/handle/1887/1


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NEW ROLE OF SIGNAL PEPT IDE

PEPTI DASE TO LIBE RATE

CTERMINAL PEPTIDESFOR MHC CLASSI PRESENTATION

Cláudia C. Oliveira1,4, Bianca Querido1,Marjolein Sluijter1, Anne F. de Groot1,Reno van der Zee1, Martijn J.W.E. Rabelink 2,Rob C. Hoeben2, Ferry Ossendorp3,Sjoerd H. van der Burg 1 and Torbald van Hall1

1Deparmen o Clinical Oncology,2Deparmen o Molecular Cell Biologyand 3Deparmen o Immunohemaology and Blood ransusion,Leiden Universiy Medical Cener, Leiden, he Neherlands;4Graduae Program in Areas o Basic and Applied Biology,Poro, Porugal.

Acceped or publicaion in: Journal of Immunology

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ABSTRACT

e signal pepide pepidase (SPP) is an inra-membrane cleaving asparyl proease

involved in release o leader pepide remnans rom he E membrane, hence is name.

We now ound a new aciviy o SPP ha mediaes liberaion o C-erminal pepides. Inour search or novel proeolyic enzymes involved in major hisocompaibiliy complex

class I (MHC-I) presenaion, we ound ha SPP generaes he C-erminal pepide-

epiope o a ceramide synhase. e display o his immunogenic pepide/MHC-I

complex a he cell surace was independen o convenional processing componens likeproeasome and pepide ransporer AP. Absence o AP aciviy even increased he

MHC-I presenaion o his anigen. Muagenesis sudies revealed he crucial role o he

C-erminal locaion o he epiope and ‘helix-breaking’ residues in he ransmembrane

region jus upsream o he pepide, indicaing ha SPP direcly liberaed he minimal

9-mer pepide. Moreover, silencing o SPP and is amily member SPPL2a led o a generalreducion o surace pepide/MHC-I complexes, underlining he involvemen o hese

enzymes in anigen processing and presenaion.

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INTRODUCTION

All nucleaed cells o our body display a represenaive selecion o he cellular proeome

in Major Hisocompaibiliy class I (MHC-I) molecules a heir suraces. is wide

array o pepides serves as ligands or CD8+

-cells or cellular immuniy. e processo pepide presenaion sars wih he proeolysis o aged or misolded proeins, a

process mainly execued by he muli-caalyic acion o proeasomes. e generaed

pepide sequences are ranslocaed rom he cyosol ino he E by he AP pepide

ransporers, where hey are assembled wih differen MHC-I molecules wih help rom

members o he pepide loading complex, including calreiculin, apasin and Ep57.

Sable pepide/MHC-I complexes are roued o he cell surace. is proeasome-AP

pahway is considered as he convenional processing roue, used o generae and shutle

he majoriy o pepides1-3. Addiional players, like ripepidyl pepidase II (PPII) and

nardilysin are responsible o urher shoren proeasomal inermediaes or, someimes, odirecly generae minimal pepide sequences ha fi in MHC-I molecules4, 5.

Alhough hese cenral elemens o he convenional anigen processing pahway

are imporan or he mainsream o pepides, subsanial MHC-I anigen presenaion

is sill deecable on cells in which he uncion o key componens is compromised.

For example, deficiency in he pepide ransporer AP, which can be regarded as a

botleneck in he pahway, resuls in a decreased surace MHC-I, bu he residual pepide

reperoire is sufficien o generae a diverse CD8+ cell subse in mice6-8 and man9, 10.

Imporanly, his cell reperoire is capable o conrol common viral inecions and rom

some AP-deficien paiens Cαβ+

CD8+

-cell clones were isolaed ha recognizedAP-independen viral anigens11, 12. ereore, AP-independen processing pahways

may sufficienly compensae or he loss o he convenional roue in order o selec a

uncional CD8+ -cell reperoire in hese paiens.

is reasoning is in line wih our recen findings on a novel caegory o cell epiopes

presened by cancer cells wih AP-deecs. We showed ha deficiency in he expression

o he pepide ransporer AP, a eaure requenly ound in human cancers, leads o he

presenaion o a broad reperoire o immunogenic pepides by MHC-I13-17. Biochemical

characerizaion o pepide reperoires rom AP-deficien cells and heir direc proficien

counerpars indeed revealed unique complemening pools o pepides14, 18, 19.wo pahways or AP-independen pepide loading are described hus ar 20. One

o hese is acive in he secreory roue o he Golgi and is mediaed by he proein

converase urin21-23. e oher concerns N-erminal leader sequences, which are

liberaed afer arrival in he E by he signal pepidase (SP). Cleavage by SP leaves small

ransmembrane leader pepide remnans in he E membrane, which are removed by he

inramembrane-cleaving signal pepide pepidase (SPP). e E-direcing pars o hese

pepides end up in he lumen o he E and can be loaded uno MHC-I molecules24, 25. An

immunogenic human umor anigen rom he leader sequence o calcionin is released by

his means and AP is dispensable or is presenaion on cancer cell lines26, 27.

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In he curren sudy we reveal he processing pahway o a C-erminal pepide rom he

ceramide synhase rh4 (also known as CerS5). is proein resides in E membranes and

docks is C-erminal epiope-conaining ail ino he bilayer. Inramembrane proeolysis

by SPP jus in ron o he epiope resuled in liberaion and AP-independen loading on

D b molecules. is represens a novel role or SPP, acing independenly rom he signal

pepidase (SP). Our daa indicae ha SPP is able o direcly liberae minimal pepide-

epiopes rom he C-erminus o proeins by inramembrane proeolysis and underlines

he involvemen o his enzyme in anigen processing.

RESULTS

The TAP-independent pro cessing pat hway of the Trh4-derived peptide

is common and conserved

e rh4-derived pepide MCLMAVM is presened by he MHC class I moleculeH-2D b on AP-deficien umor cells17. We were ineresed in he naure o is AP-

independen processing pahway ha leads o he generaion and surace presenaion o

he rh4 pepide in D b , and hereore, analyzed he generaliy o his anigen processing

pahway in differen mouse and human umor cell lines (Fig 1). Four mouse umor cell

lines o differen origin and conaining a AP deec (a lymphoma, a fibrosarcoma, a colon

carcinoma and a melanoma) were analyzed or recogniion by a previously esablished

MCLMAVM-specific CD8+ -cell clone. ese cells efficienly recognized all AP-

deficien umors, irrespecive o heir issue origin (Fig 1A). Imporanly, he differen

mechanisms underlying he AP impedimen in hese umors, being AP2 muaion (inMA-S), AP1 gene loss (in MCA), expression o a small viral inhibior (in MC38) or

AP2 gene loss (in B78H1), all led o efficien presenaion o he rh4-derived pepide,

indicaing ha presenaion o he rh4 pepide is relaed o AP-deficiency as such and

no o a specific molecular mechanism. e ac ha AP-posiive counerpar umors

presened much lower rh4 pepide a heir cell surace was no surprising, since we

previously showed ha resoraion o AP uncion decreases rh4/D b complexes a he

cell surace, mos likely due o compeiion28. ereore, he rh4 pepide presenaion is

AP-independen and operaes in differen umor ypes.

e degree o CL recogniion o he umor cell lines varied and we wondered i hese

differences could be explained by rh4 expression levels. rh4 ranscrips were quanified

by qPC and indeed revealed higher expression in he lymphoma MA-S compared o

he solid umors (Fig 1B). Expression o a splice varian o rh4, which does no conain

he D b-binding pepide, showed less variey in he umor panel. We concluded ha surace

display o rh4/D b complexes is deermined by AP uncion, rh4 proein levels and

possibly oher acors like he proease responsible or pepide liberaion.

We also waned o know i human cells were equipped wih he AP-independen

processing pahway. e mouse rh4 gene was inroduced ino he human cell lines

HeLa and HEK293, ogeher wih he mouse MHC class I gene H-2D b ha presens

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0

5

10

15

0

5

10

15

20

0

10

20

30

40

R M A . T r h 4

T r h 4 + D b

c o n t r o l

T r h 4 + D b

c o n t r o l

I F N γ

r e l e a s e ( n

g / m l )

I F N γ

r e l e a s e ( n g / m l )

RMA

lymphoma

MCA

fibrosarcoma

MC38

colon

carcinoma

B78H1.Db

melanoma

HeLa 293T

TAP status: - - - -+ + + +

N o r m a l i z e d f o l d e x p r e s s i o n

A

B C

R M A

M C A

M C 3

8

B 7 8 H

1 . D b

0

2

4

6

8

R M A

M C A

M C 3

8

B 7 8 H

1 . D

b

0.0

0.5

1.0

1.5

2.0

2.5

Trh4 Trh4 splice variant

Figure 1. Te rh4 processing pathway is conserved and broadly active. (A) Presenaion orh4-derived pepide by MHC-I was evaluaed on lymphoma (MA), fibrosarcoma (MCA), coloncarcinoma (MC38) and melanoma cells (B78H1.D b). Acivaion o rh4-specific cells is enhanced byhe absence on pepide ransporer AP. (B) mNA expression o rh4 and a naural rh4 splice varian

was deermined by quaniaive PC. e splice varian does no encode he 4 pepide sequence dueo a rame-shif. One ou o hree comparable experimens is shown. (C) Human 293 and HeLa cells

were ranseced wih mouse rh4 and H-2D b

genes and used as arges. Mean and sandard deviaion oriplicaes are shown rom one ou o hree experimens.

his pepide. e rh4-derived pepide was efficienly presened a he cell surace o boh human lines, indicaing ha he involved processing pahway was indeed also acive

in human cells (Fig 1C). Alhough hese human cells have inac AP uncion and

hereby impede rh4 pepide presenaion, he high expression levels o rh4 proein,

rom srong heerologous promoers overcomes he compeiion o pepides rom heproeasome-AP pahway, as illusraed by efficien CL recogniion o AP-posiive

MA lymphoma cells in which rh4 is expressed eigh hundred imes higher han he

endogenous level28. ogeher, hese daa show ha he AP-independen processing

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pahway ha generaes he rh4 pepide is a common and conserved mechanism

operaional in mouse and human umor cell lines o differen hisological origins.

The Trh4 protein is localized in the membrane of the endoplasmic reticulum

o unravel he processing pahway o he rh4-derived pepide, we nex deermined hecellular localizaion he rh4 proein. e rh4 proein (also known as CerS5) belongs o

a amily o ceramide synhases (CerS1 o CerS6) and exiss in wo isoorms as a resul o

alernaive splicing o he ranscrip17. e shorer proein varian (UniPro Q9D6K9-2)

conains he D b-presened pepide MCLMAVM a he complee end o is C-erminus.

e longer proein (UniPro Q9D6K9-1) lacks his pepide sequence due o a rame shif

a he las inron-exon boundary 17. Alhough he longer isoorm was described o be a

muli-ransmembrane spanning proein in he endoplasmic reiculum32 , no daa were

available concerning he rh4 varian comprising he pepide-epiope. Algorihm programs

predicing opology o ransmembrane proeins anicipaed ha he shor rh4 proein would ransgress membranes seven imes wih he C-erminal amino acids 373-384 locaed

wihin he lipid bilayer, direced owards he lumen (Fig 2A). e V5 anibody epiope was

inroduced a wo disinc sies o rh4 gene consrucs (C-erminal afer he epiope and

N-erminal a amino acid 32) and subcellular localizaion sudies were perormed using

conocal microscopy. We observed a srong and clear co-localizaion wih E markers, bu

no wih he Golgi wih boh consrucs (Fig 2B or N-erminal ag and Suppl Fig 1A or

C-erminal ag). E co-localizaion was also observed in AP-negaive cells (Suppl Fig 1B).

An inermediae degree o co-localizaion wih miochondria was ound (Fig. 2B). I is

known ha he CerS1, CerS2 and CerS6 ceramide synhases can indeed be deeced in

miochondria33 , bu, alernaively, his miochondria co-localizaion migh merely reflec

E membranes ha are in close proximiy o miochondria. ese daa indicae ha

rh4 is a mulimembrane spanning proein in E membranes and ha is C-erminal ail,

comprising he epiope, docks ino his membrane direcing owards he E lumen.

Inhibitors of intramembranous aspartyl proteases prevent peptide liberation

Nex, we invesigaed which amily o proeases was responsible or he proeolyic

cleavage o rh4 resuling in liberaion o he C-erminal pepide. An array o inhibiors

blocking hydrolysis by differen classes o proeases were applied in a recovery assayin which AP-deficien MA-S cells were briefly acid sripped o remove cell surace

MHC-I and allow or re-emergence o rh4/D b pepide complexes. Complee recovery

o rh4/D b was reached wihin six hours, as deermined wih a pepide-specific cell

clone (Fig 3A). is highlighed a coninuous and efficien supply o rh4 pepide rom

his alernaive processing pahway. wo enzymes were likely candidaes, since hey were

described o resul in AP-independen pepide loading: signal pepidase and urin20, 21.

However, he specific inhibiors DCI and Dec-VK-CMK, respecively, ailed o impair

he generaion o rh4/D b complexes (Fig 3B), indicaing ha rh4 processing is mediaed

by a novel mechanism. In line wih our previous daa, inhibiion o he proeasome raher

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G o l g i

M i t o c h o n d r i a

E R

T r h 4 ( a n t i - V 5 ) B I P

G i a n t i n

M i t o f i l i n

T r h 4 ( a

n t i - V 5 )

O r g a n e l l e s t a i n i n g

M e r g e

C o

- l o c a l i z a t i o n

7 0 . 9 4 %

9 . 0 2 %

4 6 . 6 5 %

C y t o s o l

L

u m e n

N ’

C

’

T r h 4

e p i t o p

e

V 5 t a g

A

B

F i g u r e 2 . T e C - t e r m i n a l t a i l o f r h 4 i s l o c a l i z e d w i t h i n t h e E R m e m b r a

n e . ( A ) P r e d i c t i o n o t h e r h 4 t o p o l o g y i n m e m b r a n e s a c c o r d i n g t o a l g o

r i t h m s o f w a r e

( w w

w . p r e d i c t p r o t e i n . o r g ) . e C - t e r m i n a l p e p t i d e - e p i t o p e i s i n d i c a t e d i n r e d . ( B ) C o n o c a l m i c r o s c o p y i m a g i n g o V 5 - t a g a t t h e N - t e r m i n u s o r h 4 p r o t e i n i n

c o m

b i n a t i o n w i t h s t a i n i n g o r e n d o p l a s m i c r e t i c u l u m ( B I P ) , G o l g i ( g i a n t i n ) o r m i t o c h o n d r i a ( m i t o fi l i n ) . M e a

n p e r c e n t a g e s o c o l o c a l i z a t i o n w e r e c a l c u l a t e d o n

t e n

i m a g e s o a t l e a s t t h r e e i n d e p e n d e n t e x p e r i m e n t s .

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% r

e c o v e r y

T r h

4 / D b

0 1 2 543 6

n o

t r e a

t m e n

t

c o n

t r o

l

time after stripping (h)

0

20

40

60

80

100

120

S t r i p

E p o x o m

i c i n

D C I

F u r i n

i n h i b i t o r

0

25

50

75

100

125

150

C T L r e c o g n

i t i o n

( % )

A B

F

C D

G

0 20 40 60 80 1 00 120

CTL recognition (%) CTL recognition (%)

S t r i p

c a

l p e p

t i n

c a

l p a

i n i n h

. I V

c a

l p a s

t a t i n

P D 1 5 1 7 4 6

P D 1 5 0 6 0 6

0

25

50

75

100

125

150

S t r i p

B u

t a b i n d i d e o x a

l a t e

C a p

t o p r i

l

L e u p e p

t i n

( z - L

L ) 2 - k e

t o n e

P h e n a n

t h r o

l i n e

A p r o

t i n

i n

D A P T

B e s

t a t i n

z - v a

d - f m

k

L e u c

i n e

t h i o l

0

25

50

75

100

125

150

(z-LL)2-ketone DAPT

0.01 µM

0.1 µM

1 µM

10 µM

Stripping

2.5 µM

10 µM

40 µM

Stripping

C T L r e c o g n i t i o n ( % )

0 20 40 60 80 100 120

S t r i p

D A P T

c o n t r o l

0

20

40

60

80

100

120

( z - L L ) 2 - k e t o n e

E

Figure 3. Te Db-presentation of the rh4-derived peptide is generated by intramembrane-cleavingaspartyl proteases. (A) ecovery kineics o rh4/D b complexes on AP-deficien MA-S cells afer

brie sripping wih mild acid buffer. Cells were esed wih rh4-specific cell clone in inracellularIFN-γ saining. (B) ecovery efficiency o rh4/D b complexes was esed in he presence o an array oproease inhibiors. Inhibiors o proeasome and enzymes known o be involved in AP-independenprocessing pahways, (C) inhibiors o calpain amily, (D) all oher esed inhibiors. (E) AP-deficienfibrosarcoma MCA cells were esed wih inhibiors or asparyl proeases. (F) Concenraion range ohe uncional inhibior (z-LL)

2-keone and (G) DAP on MA-S cells. rh4-specific cells (filled

bars) were used and conrol cells (open bars) ha recognize an independen K b-presened pepide

on MA-S. All panels show compiled means and sandard deviaions rom a leas hree independenexperimens, seting he recovered cells a 100%.

increased rh4/D b complexes (Fig 3B), suggesing ha he proeasome somehow acs asa compeior or he novel processing mechanism28.

Nex, we observed ha he common calpain inhibior calpepin parly decreased rh4

presenaion a relaively high concenraions (> 20 uM) (Fig 3C and Suppl Fig 2A). e

main arges or calpepin are calpain 1 and calpain 2. We deermined he expression ohese wo calpain members in MA-S cells and observed ha calpain 2 was expressed

bu calpain 1 was no deecable (Suppl Fig 2B). ereore, we silenced he expression o

calpain 2, bu his did no lead o reduced rh4/D b pepide complexes a he cell surace

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(Suppl Fig 2C-D). Furhermore, more selecive inhibiors or he calpain amily did no

affec he CL response, collecively suggesing ha he marginal blocking effec by

calpepin was based on cross-reaciviy o oher proeases (Fig 3C).

We hen coninued he screen wih several oher classes o chemical inhibiors argeing

serine-, cyseine-, asparyl- and meallo-proeases, and aminopepidases (Fig 3D). e

resuls revealed wo inhibiors or asparyl proeases, (z-LL)2-keone and DAP, ha

srongly decreased presenaion o he rh4-derived pepide. Imporanly, none o he oher

eigh proease inhibiors influenced he rh4/D b presenaion, suggesing ha he involved

asparyl proease was necessary and sufficien. e inhibiory effec was also observed when

we esed fibrosarcoma cells, indicaing ha he inhibiion was no cell-ype resriced (Fig

3E). iraion o he wo acive compounds demonsraed a 50% decrease a concenraions

in he lower range in his cellular assay, 300 nM or (z-LL)2-keone and 5 uM or DAP (Fig

3F and G). ese concenraions were clearly no oxic or he arge cells and, moreover,

recogniion o reaed MA-S cells by conrol cell clones specific or anoher pepide

epiope was unalered, excluding a general deec in he MHC class-I presenaion capaciy

by (z-LL)2-keone and DAP (Fig 3F and G). ese wo compounds were known o arge

asparic acid proeases ha are locaed wihin membranes, paricularly members o he

signal pepide pepidase (SPP) amily 34, 35. Ineresingly, calpain inhibiors were shown o

block he acion o SPP a higher concenraions, corroboraing our findings in he screen

wih calpepin (Fig 3C)36. ogeher, hese resuls le us conclude ha he liberaion o he

C-erminal rh4 pepide wihin he E membrane is par o a novel processing pahway or

AP-independen pepides and is disinc rom he previously described roues.Signal Peptide Peptidase is responsible for the processing of

the C-terminal peptide

e SPP amily is also known as inramembrane-cleaving proeases (I-CLiPs) o he

asparyl ype and his amily is involved in many biological processes, including cell

prolieraion, differeniaion and immuniy 34, 37, 38. SPP and SPPL3 were repored o

be localized in E membranes, orming caalyic caviies in which hydrolysis can ake

place wihin membranes39. We firs analyzed he expression o he amily members a

mNA level in he umor cell lines acive in rh4 pepide processing (Fig 4A). All amily

members were expressed o varying degree in his cell panel, excep SPPL2c, which was

hardly deecable. High levels o SPP were measured in all cells. Differences in expression

levels in he umor cell panel promped us o profile ranscrips o hese genes in normal

mouse issues, especially since his inormaion is lacking in he curren lieraure (Suppl

Fig 2E). NA exracs rom whole mouse organs were analyzed or hese inramembrane

asparic pepidases and a general low expression or SPPL2b and SPPL2c was ound.

Similar expression profiles were seen or SPP, SPPL2a and SPPL3 in ha hymus and

brain conained low ranscrip numbers and lung was high (Suppl Fig 2E).

Nex, we waned o deermine which amily member was responsible or he liberaion

o he C-erminal rh4 pepide in umor cells and silenced each gene individually using

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shor hairpin NA consrucs. SPPL2c was no included in his analysis, since i was no

expressed in he umor cells (Fig 4A). Efficiency o gene silencing was approximaely

50-60% or all genes and, imporanly, he shNA consrucs were specific in ha he

oher members were no argeed (Fig 4B). We hen used his cell panel o deermined

effecs on recogniion by rh4-specific CL. Downregulaion o SPP clearly decreased

he recogniion by hese CL (Fig 4C). Similar decrease o rh4 pepide presenaion

was observed in AP-negaive MCA fibrosarcoma cells (Suppl Fig 2F-G). Silencing

o he oher SPP amily members resuled in a marginal variaion o CL recogniion

(Fig 4C). e presenaion o he rh4 pepide was no compleely blocked by SPP

silencing, mos likely due o a subsanial residual expression o he proease. e residual

CL recogniion could urhermore be blocked by (z-LL)2-keone, supporing his

noion. Imporanly, an independen oher pepide-epiope presened by MA-S was

no affeced by SPP silencing (Fig 4D). ese daa and he ac ha SPP is expressed

in all mouse cells ha naurally display rh4/D b complexes and he E localizaion o

rh4 subsaniae he conclusion ha he SPP enzyme is crucial or he processing o he

C-erminal pepide-epiope o rh4 in a AP-independen way.

The transmembrane region directly upstream of the Trh4 peptide is

crucial for Trh4 processing

o sudy he underlying mechanism o rh4 pepide processing in deail and o deermine

criical regions or he proeolyic process, exensive muagenesis analysis o rh4 was

perormed. Firs, some larger segmens a he N- and C-erminus o he proein were

deleed. emoval o he firs 32 aa did no diminish he processing efficiency o he pepide-epiope (Fig 5A), whereas we hypohesized ha his region uncioned as a leader sequence

rouing he proein o he E, as indeed was prediced by he online SignalP 4.1 Server

(www.cbs.du.dk). Conocal microscopy analysis however confirmed ha his consruc

was sill localized in E (Suppl Fig 1C). We hen removed segmens locaed near he

C-erminus, saring wih he sequence corresponding o amino-acids 340-374, which is a

cyosolic loop locaed beween he wo las ransmembrane domains (see Fig 2A). We did a

progressive deleion in his consruc comprising he upsream amino-acids 315-330, which

includes mos o he second las ransmembrane domain. However, none o hese large

deleions decreased rh4 pepide processing (Fig 5A). In order o exclude he possibiliy

ha hese alered consrucs delivered deecive and hus proeasome-argeed proeins,

we confirmed ha he rh4 pepide presenaion rom hese muan consrucs was sill

(z-LL)2-keone and DAP dependen (Fig 5B). en, nine amino-acids jus upsream o

he C-erminal pepide were removed. is resuled in a more han 60% impairmen o

CL recogniion (Fig 5A), indicaing ha his inramembrane srech, jus upsream o he

pepide-epiope was criical or SPP cleavage. Furhermore, he C-erminus was exended

by cloning small V5-conaining ags o 14 and 47 amino-acids. Alhough hese proeins

were properly expressed, as esed by wesern blo (Suppl Fig 3A), and were localized in he

E (Suppl Fig 1A), hey did no resul in any rh4 pepide presenaion (Fig 5A), implying

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0

200

400

600

800

1000

N o r m a l i z e d f o l d

e x p r e s s i o n

SPP SPPL2a SPPL2b SPPL2c SPPL3

RMA-S

MCA

MC38

B78H1.Db

A

B

shRNA-SPP

shRNA-SPPL3

empty vector

shRNA-SPPL2a

shRNA-SPPL2b

C

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

0

5

10

15

I F N γ


number of targets

0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5

0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5

shRNA-SPP

shRNA-SPPL2a

shRNA-SPPL2b

shRNA-SPPL3

empty vector

SPP SPPL2a

SPPL2b SPPL3

Normalized fold expression Normalized fold expression

D

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

0

10

20

30

40

number of targets

shRNA-SPP

shRNA-SPPL2a

shRNA-SPPL2b

shRNA-SPPL3

empty vector

Figure 4. Te intramembrane-cleaving signal peptide peptidase is involved in rh4 processing.(A) mNA expression o SPP , SPPL2a , SPPL2b , SPPL2c and SPPL3 genes was deermined by qPCin umor cell panel. (B) Silencing o SPP amily members in MA-S cells was reached by leniviralranser o shNA consrucs. Empy shNA vecor served as conrol. Efficiency and specificiy odownregulaed levels is shown as measured by qPC. (C-D) Cell panel wih silenced single SPP amilymembers was esed or recogniion by rh4-specific cells (C) or conrol cells (D) reacive o heindependen K b-presened pepide mi3 by MA-S. Mean and sandard deviaion rom one ou o hree

experimens is shown. Difference beween SPP and conrol vecor is saisically significan (p


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wt

Del #1

Del #2

Del #3

Del #4

Ext. #1

Ext. #2

Construct

ACTL

recognition

379 3871

2 32

340 374

3 15 3 30

369377

1 14

1 47

Trh4 core protein CTL epitope Deletion Extension

+++

++

++

++

+

-

-

SequenceCTL

recognition (%)MutationsSDM

construct

wt 100...WWQLDSSILC MCLRMTAVM

# 1 aa375 106...WWQLDSI ILC MCLRMTAVM

# 6 aa377 82...WWQLDSSI A C MCLRMTAVM

# 3 aa378 62...WWQLDSSILG MCLRMTAVM

# 5 aa374/375/378 109...WWQLDII ILG MCLRMTAVM

# 2 aa374-375 99...WWQLDII ILC MCLRMTAVM

# 7 aa376-377 38...WWQLDSS AA C MCLRMTAVM

# 8 aa376-378 27...WWQLDSS AAG MCLRMTAVM

# 9 aa375-378 18...WWQLDSIAAG MCLRMTAVM

# 10 aa374-378 14...WWQLDIIAAG MCLRMTAVM

# 4 aa378 9...WWQLDSSILI MCLRMTAVM

- 3 6 9

- 3 8 0

- 3 7 9

- 3 7 8

- 3 7 6

- 3 7 7

- 3 7 5

- 3 7 4

- 3 7 3

- 3 7 2

- 3 7 1

- 3 7 0

- 3 8 7

- 3 8 6

- 3 8 5

- 3 8 4

- 3 8 3

- 3 8 2

- 3 8 1

C

Name

0

10

20

30

40

50

wt Del #1 Del #2 Del #3

B

% I

F N γ

+ T

- c e l l s

I F N γ


D

1 6 0

8 0 0

4 0 0 0

2 0 0 0 0

0

5

10

15

20

wt

SDM #5

SDM #4

SDM #3

SDM #2

number of targets

2(z-LL) -ketone

control

DAPT

ha he C-erminal locaion o he pepide in he rh4 proein is crucial or is liberaion.Failure o process he rh4 pepide when i was no a he complee C-erminus migh be

relaed o he absence o carboxyl-pepidases in he E and suggess ha he SPP cleavage

producs do no ravel hrough he cyosol. Furhermore, we also esed i he C-erminal

region would uncion as a leader sequence or he rh4 proein, bu removal o he las 20amino-acids (aa368-387) did no change is subcellular localizaion (Supp Fig 1D). We

hereore concluded ha rh4 pepide is direcly liberaed by SPP rom he maure proein

afer which he pepide is available in E or loading ino MHC-I molecules.

Figure 5. Te intramembrane region directly upstream of the rh4 peptide is crucial for processing.(A-B) rh4 consrucs wih deleions or exensions were esed or liberaion and MHC-I presenaion ohe C-erminal pepide-epiope (aa379-387). (A) DNA plasmid consrucs were ransienly ransecedino HeLa cells ogeher wih he mouse D b gene. Exend o IFN-γ release by cells was quanifiedand scored as “+++” (wild-ype consruc, 100%), “++” (50-90%), “+” (10-50%) and “-“ (


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e imporance o he direc upsream amino-acids o he rh4 pepide suggesed ha

he SPP cleavage sie was locaed wihin his las membrane region (aa374-387). Single

amino acid subsiuions were inroduced using sie-direced muagenesis o deermine

he precise requiremens or cleavage by SPP. Several publicaions demonsraed he srong

preerence o SPP or subsraes wih helix-desabilizing residues in heir ransmembrane

domain40-42. Amino-acids like asparagine, serine and cyseine disurb a perec alpha-

helical conormaion o he ransmembrane domain and are hereore reerred o as

‘helix-bending’ or ‘helix-breaking’ residues. ree o such amino-acids are presen in he

rh4 membrane region: wo serines (aa374 and 375) and one cyseine (aa378) (Fig 5C).

Firs, he wo serines were replaced by isoleucines, which srongly conribue o a perec

alpha-helical srucure. In conras o our expecaions, hese muaions did no affec rh4

presenaion a all (Fig 5C and D; SDM #2). Second, he cyseine residue (aa378) in ron

o he epiope was subsiued or a glycine (SDM #3), which sill has some helix-breaking

capaciy, or an isoleucine (SDM #4), which removes he helix-breaking naure. ese

aleraions had a very srong impac on pepide liberaion and he C378I consruc nearly

compleely ailed o produce he rh4 pepide. ird, we combined he cyseine muaion

C378G wih he serine muaions and surprisingly ound ha inroducion o wo

isoleucines improved he rh4 processing (Fig 5C and D; SDM #5), indicaing ha he

more upsream serine residues were acually counerproducive or opimal SPP cleavage

and suppor he imporance o he cyseine residue direcly upsream o he epiope.

Fourh, we progressively subsiued he complee srech o amino-acids in his region also

using alanines which are considered o promoe helix-desabilizaion

43

. Collecively, heseconsrucs displayed a clear gradual decrease in pepide presenaion (Fig 5C and Suppl

Fig 3B). ogeher, hese daa poin o he imporance o he cyseine direcly upron o he

epiope, mos likely marking he acual SPP cleavage sie and sugges ha nearby flanking

residues indirecly influence his process via conormaional change in he inramembrane

alpha-helix. In line wih his hypohesis is he finding ha presenaion o he rh4 pepide

does no depend on he N-erminal rimming pepidase ERAP (Fig 3D), suggesing ha

he minimal 9-mer epiope is direcly generaed by SPP cleavage wihou urher need

or C-erminal nor N-erminal rimming. e C-erminal liberaion o his CL pepide-

epiope by he asparyl proease SPP wihin he E membrane exemplifies a novel way by which AP-deficien cells are sill able o presen a diverse anigen reperoire.

Contribution of SPP protease family to MHC-I peptide repertoire of TAP-

negative cells

We urher examined he conribuion o he SPP amily o proeases o he overall

MHC-I presenaion o AP-deficien cells. We analyzed he surace display o he

wo alleles o his mouse srain, D b and K b , on MA-S cells in which individual SPP

members were silenced by shNAs. SPP and SPPL2a silencing resuled in reducion o

K b molecules o 77% and 65% o conrol, respecively (Fig 6). e presenaion o D b

molecules on hese cells and MHC-I surace display on MA-S cells in which SPPL2b

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cleavage produc is unlikely, since inhibiors o amino-pepidases did no preven rh4

pepide presenaion and he cyseine jus upron o he 9-mer epiope appeared o be

criical in he SPP cleavage process (see Fig 3 and 5C). Moreover, direc release o he

liberaed pepide ino he E lumen is very likely, due o he ype II ransmembrane

orienaion o he rh4 proein ail (see Fig 2A) and he ac ha he epiope is locaed

a he very C-erminal end o he proein. e incompeence o C-erminally exended

versions o rh4 o presen he pepide (see Fig 5A) corroborae he idea ha he pepide

is direcly released in he E lumen where i needs a ree C-erminus or loading ino

MHC-I molecules. e exac pepide loading mechanism o he rh4 membrane pepide

however remains o be deermined.

e involvemen o SPP in he C-erminal processing o a ransmembrane proein

iniially surprised us. e aciviy o SPP was hus ar largely relaed o he removal o small

runks o signal-sequence-derived ransmembrane pepides rom he E membrane34, 37, 42.

e processing o leaders rom nascen proeins requires a firs cu by signal pepidase

(SP), afer which he remaining ransmembrane srech is cu in wo pars by SPP.

However, he liberaion o he rh4 pepide did no require SP aciviy, since is inhibior

DCI did no preven he presenaion o he epiope. Furhermore, he rh4-pepide is

no par o a signal sequence and removal o he N- or C-erminal domain did no roue

he rh4 proein o a differen subcellular localizaion (see Suppl Fig 1C-D). Neverheless,

he involvemen o SPP wih rh4 cleavage fis wih he srong preerence or ype II

membrane segmens and wih he ‘helix-breaking’ residues ha are ofen ound in SPP

arge proeins25, 41, 42. ereore i seems ha he role o SPP is more exended han he

release o ransmembrane leader-sequences orm he E membrane. Ineresingly, i has

been suggesed ha SPP is physically associaed wih misolded membrane proeins44, 45

and as such migh uncion as a chaperone o dispose membrane aggregaes.

e rh4-derived pepide is an example o AP-independenly processed pepides.

is paricular SPP-mediaed processing pahway is operaional in AP-negaive as well

as AP-posiive cells (see Fig 1) and also in umor as well as healhy cells 28. ereore,

he AP-independen pahway described here seems o be par o he “normal aciviy”

o cells and does no represen a disease-relaed eaure. Over he years, some oher AP-

independen pahways have been characerized. ey comprise pepides generaed in he

secreory and vesicular comparmens by he proein converase urin22, 23. Second, leader

sequence derived pepides are requenly AP-independenly processed24. Pepides

ha arrive in he E via his way overcome he need o AP ranspor. Ineresingly,

AP-independen processing was observed earlier using several C-erminally agged

consrucs o E-argeed proeins, eeding he speculaion on a common ‘C-erminal’

processing rule46, 47. We hypohesize ha SPP migh be a cenral player or his caegory

o AP-independen anigen processing.

e amily o inramembrane-cleaving asparyl proeases (SPP amily and presenilins)

have imporan biological roles in signal ransducion, cell differeniaion and immuniy

25, 48

.For insance, SPPL2a and SPPL2b were shown o be responsible or he release o he

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NF-α inracellular domain, leading o expression o he pro-inflammaory cyokine IL-12

in dendriic cells38 and also or he release o he inracellular domain o asL in cells49.

ese proeases are mainly locaed in he vesicular pahway and a he cell surace. ecenly,

SPPL2a was repored o be involved in he proeolysis o he invarian chain (CD74) and

hereby criical or he developmen and survival o B-cells and DCs50-52 , maybe explaining

he effec we ound on MHC class I expression (Fig 6). We now exend on he involvemen

o SPP amily members in immuniy by showing heir role in alernaive pahways o

MHC-I anigen processing, hereby enabling cell immune responses.

MATERIAL AN D METHODS

Cell lines

e mouse umor cell lines MA, MA-S (AP2-deficien), MCA (AP1-deficien), MC38,

B78H1 (AP2-deficien) and human HeLa and 293 have been described previously 14, 17, 28.

Where indicaed, cell lines were ranseced wih H-2D b or rh4 genes (accession numbers:

gene BC043059 (www.ncbi.nlm.nih.gov/nuccore), proein UniProKB Q9D6K9-2 (www.

unipro.org/unipro) via reroviral ransducion using he LZS vecor conaining GFP

behind an inernal ribosome enry sie16. e mouse AP genes were inroduced ino MCA

and B78H1 cells by gene ranser. MC38.UL49.5 cells conain he UL49.5 gene rom he

Bovine Herpes Virus-1 which blocks mouse AP aciviy 16, 29.

Generaion and culure o CD8+ cell clones used in his sudy were previously

described: D b-resriced CL clone B5 specific or he AP-independen rh4 pepide

(MCLMAVM); K b-resriced CL clone mi3 specific or a ye unknown EIPP

pepide and he D b-resriced CL clone 1 specific or he AP-dependen MuLV gag-

leader pepide (CCLCLVFL)16, 17, 30.

All cells were culured in complee IMDM medium (Invirogen, Carlsbad, CA)

conaining 8% hea-inacivaed FCS, 100 U/ml penicillin, 100 μg/ml srepomycin

(Lie echnologies, ockville, MD), 2mM L-gluamine (Invirogen) and 30 μmol/L o

2-mercapoehanol (Merck, NJ, USA) a 37°C in humidified air wih 5% CO2.

Protease inhibitor experiments

Cells were resuspended in X-vivo medium (Lonza) supplemened wih 0,5% o BSAand incubaed wih proease inhibiors or 1h a 37°C, under coninuous mixing. Cells

were incubaed or 2-4 min wih mild acid cirae/phosphae buffer (pH 3.1) a room

emperaure o disrup MHC class I/pepide complexes28. Cells were washed wice and

incubaed wih proease inhibiors or addiional 6h under he condiions menioned

above and hen reaed wih breeldin A. Inhibior-reaed cells were hen incubaed wih

he indicaed cell clones or 18h in he presence o breeldin A and inracellular sained

or IFNγ producion28. Chemical inhibiors used in hese assays are: epoxomicin (1µM,

Sigma-aldrich), 3,4-Dichloroisocoumarin (5µM, Sigma-aldrich), Decanoyl-VK-CMK

(10µM, Merck), calpepin (30µM, Merck), calpain inhibior IV (13µM, Merck), calpasain

62


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able I. Primer sequences for quantitative PCR for the mouse SPP family.

Name Gene ID Forward primer (5’->3’) Reverse primer (5’->3’) Size (bp)

SPP 14950 CAGGACGCGGAGAAG GAAGAGGGGCGGT 138SPPL2a 66552 CGAGGCAAAGGAACAAG GAGGAAGGAATAGGACAC 133SPPL2b 73218 CATGCCTCGCCGA GAGCGTGCCACCT 142SPPL2c 237958 GCATGCCCGCGGGGC GGGCCCCGGACCGGG 181

SPPL3 74585 CGCAGACAACACAAGA GCAGGGAAGAGGAGAC 93

pepide (10µM, Merck), PD151746 (50µM, Sana Cruz Biohecnology), PD150606(50µM, Sana Cruz Biohecnology), buabindide oxalae (400µM, OCIS Bioscience),

capopril (100µM, Sigma-aldrich), leupepin (100µM, Merck), (z-LL)2-keone (5µM,

Merck), 1,10-Phenanhroline monohydrae (400µM, Sigma-aldrich), aproinin (10µM,

Merck), DAP (10µM, Sigma-aldrich), Besain hydrochloride (100µM, Sigma-aldrich),z-vad-mk (10µM, Merck), L-leucinehiol (60µM, Sigma-aldrich) and DL-Dihiohreiol

(5mM, Sigma-aldrich). None o he inhibiors was oxic a he applied concenraions, as

deermined by rypan-blue couning and annexin-V saining.

T-cell activation assays and flow cytometry

Inracellular cyokine saining o IFN-γ in cell clones was perormed or he proease

inhibior screen and expressed as percenage posiive cells31. IFN-γ secreion by -cellclones was measured by ELISA as previously published28. Daa shown represen mean

values obained rom riplicae es wells and error bars represen sandard deviaion ohese values. Flow cyomery analysis o surace expression o D b and K b molecules was

perormed using direcly labeled ani-D b mAb (clone 28.14.8S; BioLegend, Caliornia,USA) and ani-K b mAb (clone AF6-88.5, BioLegend). Cells were measured using a FACS

Calibur and analysed wih Flowjo sofware (ree Sar, Ashland, O).

Quantitative PCR analysis

oal NA isolaion was perormed using Neasy Mini Ki (Qiagen, Maryland, USA).

500 ng o purified oal NA was used o synhesize cDNA using High Capaciy NA-o-

cDNA Ki (Applied Biosysems, Foser Ciy, USA). Quaniaive PC on wo differenranscrips o rh4 was done using unique orward primers. Forward primer or he

rh4-epiope conaining ranscrip 5’-GCAGACCCCTACGGAAGCGC-3’ and or

he shor ranscrip 5’-ACACACGCGGCAC-3’. Common reverse primer was

5’-CGCGGCACCTAGACACCTCC-3’. e primers used o deec he expression o

mouse ranscrips rom SPP, SPPLs and calpains were designed by us using he Beacon designersofware and are lised in Supplemenary able 1 (calpain amily) and able 1 (SPP amily). All

primer ses were validaed o be specific or he cognae genes by sequencing o he amplified

PC producs. For he PC reacion SensiMix™ SYB No-OX ki rom GC Bioech Bioline

(Alphen aan den ijn, Neherlands) was used in a C1000™ ermal Cycler (Bio-ad, Hercules,CA, USA) and resuls were analysed using Bio-ad CFX manager sofware.

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Confocal microscopy

Human HeLa cells or mouse MCA cells were plaed in 12-wells plaes on glass coverslips

and ranseced he nex day wih he indicaed rh4 gene consrucs using Lipoecamine

2000 (Invirogen) according o he manuacurer’s proocol. Afer one day cells were

sained wih monoclonal anibodies: cells were fixed wih mehanol or 30 minues a

4˚C, washed wih BSB (BS wih blocking reagen rom Boehringer Mannheim) and

incubaed or 1.5 hours a room emperaure wih he firs anibody dilued in 500 μl

BSB. e anibodies used were ani-V5 (Invirogen, 960-25), ani-Gianin (Abcam,

ab24586) and ani-BIP (Abcam, ab21685). Cells were hen washed again wih BSB

and incubaed wih second anibodies; ani-rabbi-Alexa488 (Invirogen, a11008) and

ani-mouse-Alexa594 (Invirogen, a11005) or 1.5 hours a room emperaure. Finally,

cells were washed wih BSB and he coverslips were pu on microscope glass slides wih

5μl o ProLong Gold aniade reagen (Invirogen) conaining Hoechs (Enzo, Hoechs33342). e sained cells were imaged wih a CS SP5 fluorescen conocal microscope

(Leica, Germany) using LAS AF sofware. Sained samples were observed a room

emperaure. All images were acquired wih a 63X glycerol immersion objecive lens NA

1.4 (Leica). Analysis o colocalizaion was perormed wih LAS AF sofware (Leica).

shRNA treatment

MISSION® shNA consrucs specific or mouse SPP, SPPL2a, SPPL2b, SPPL3 and calpain

2 were obained rom Sigma-Aldrich and leniviral encapsulaion was reached in HEK.293

cells via ransecions wih calcium phosphae (Promega) o pCMV-VSVG, pMDLg-pE,

pSV-EV and pLKO plasmid wih puromycin resisance gene and a shNA sequence or

empy vecor. MA-S cells were incubaed wih lenivirus paricles encoding shNA and

paricles encoding GFP in a 10:1 raio or 20h a 37°C. en he cells were washed and

resuspended in complee medium supplemened wih 1µg/ml o puromycin and culured

or 4 days. GFP expressing cells were sored and used in experimens.

Trh4 gene constructs

Lengh varians o rh4 (deleions and exensions) were perormed on he cloned cDNA

(accession number BC043059) in pcDNA3.1 plasmid. C-erminal exensions were creaed

by removing he sop codon o rh4 in he pcDNA3.1/V5-His vecor (Invirogen, Lieechnologies), resuling in he in rame encoding V5 anibody epiope. e deleion varians

were based on an N-erminally agged V5 consruc in which he anibody sie was inroduced

afer nucleoide nr 96, which was supposed o be he leader sequence. We inroduced a

NO-I resricion sie a his place by creaing wo complemenary PC producs, resuling

in wo alered amino acids a posiion 32-33 (rom DL o AA). en he 14 aa long V5

sequence was inroduced a his NO-I sie using long synheic oligonucleoides including

NO-I overhangs. Orienaion and sequence o his inser was validaed by sequencing. is

‘N-erminal’ agged rh4 consruc was hen used o creae several deleion varians using

PC primer ses. All PC amplificaions were perormed wih Accuprime ki (Invirogen).

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1. Cresswell, P., Ackerman, A.L., Giodini, A., Peaper,D.. & Wearsch, P.A. Mechanisms o MHCclass I-resriced anigen processing and cross-presenaion. Immunol. Rev. 207 , 145-157 (2005).

2. ock, K.L., Faran-Arribas, D.J. & Shen, L.Proeases in MHC class I presenaion and cross-presenaion. J Immunol 184 , 9-15 (2010).

3. Vyas, J.M., Van der Veen, A.G. & Ploegh, H.L.

e known unknowns o anigen processing andpresenaion. Na Rev Immunol 8 , 607-18 (2008).

4. Kessler, J.H. e al. Anigen processing bynardilysin and hime oligopepidase generaescyooxic cell epiopes. Na Immunol 12 , 45-53 (2011).

5. Seier, U. e al. An essenial role or ripepidylpepidase in he generaion o an MHC class Iepiope. Na Immunol 4 , 375-9 (2003).

6. Aldrich, C.J. e al. Posiive selecion o sel- and

alloreacive CD8+ cells in AP-1 muan mice. Proc. Nal. Acad. Sci. USA 91 , 6525-6528 (1994).

Sie-direced muagenesis was done on he N-erminal V5 consruc in pcDNA3.1using USB Change-I Muliple Muaion Sie Direced Muagenesis echnology

(Affymerix). Generaed clones were sequenced o validae he inroduced changes.

Western blotCells were lysed in 1% Nonide P-40 lysis buffer (50 mM ris-HCl, 150 mM NaCl

(pH 8.0), 1% NP40, 1mM leupepin, 1mM 4-(2-aminoehyl)-benzene-sulonyl fluoridehydrochloride (Sigma-Aldrich,e Neherlands). e samples were mixed 1:1 wih

sample buffer (0,1 M ris (pH 8.0), 4% SDS, 20% glycerol, 10% 2-mercapoehanol, 0.05

% bromophenolblue) and hea up o 95°C or 1 minue. e samples were loaded in pre-cas

gels 4-15% (Bioad) using he Mini-POEAN® era 4-gel verical elecrophoresis

sysem (Bioad). e BenchMark Pre-Sained Proein Ladder (Invirogen) was included.Gels ran or 25 min a 250V and bloted in rans-Blo® urbo™ ranser Sysem (Bioad)

according o he manuacurer recommendaions. Afer bloting, he membrane wasincubaed overnigh a 4°C in 5% low-a milk/BS (10 mM ris-base, 150 mM NaCl,

0.05% ween 20, pH 7.4). e nex day he blos were sained wih ani-V5 monoclonalanibody 1:2500 (Invirogen) in 1% low-a milk/BS or 2 hours a room emperaure.

Afer he saining, he membrane was washed hree imes or 5 min in BS. Nex, he

membrane was incubaed wih HP-labeled goa ani-mouse Ig anibody (1:2000) in 1%

low-a milk/BS or 2 hours a room emperaure, ollowed by hree imes washing in

BS and hree imes in BS. Visualizaion o he blo was perormed using ECL Plus Wesern bloting deecion reagens (GE Healhcare) on films.

ACKNOWLEDGEMENTS

We like o acknowledge Dr Pieer van der Pol, Nicole Schlagwein and Joop Wiegan(LUMC, Neherlands) or heir help in immunofluorescen conocal microscopy. Dr.

Isaac Donkor (Memphis, ennessee, USA) kindly provided specific calpain inhibiors

and Dr amon Arens is acknowledged or criical reading o he manuscrip.

Financial suppor was received rom Poruguese Foundaion For Science and

echnology (MCES) Porugal (SFH/BD/33539/2008 o CCO).

REFERENCES

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34. Golde, .E., Wole, M.S. & Greenbaum,D.C. Signal pepide pepidases: a amily oinramembrane-cleaving proeases ha cleaveype 2 ransmembrane proeins. Semin Cell Dev Biol 20 , 225-30 (2009).

35. Weihoen, A. e al. argeing presenilin-ypeasparic proease signal pepide pepidase wihgamma-secrease inhibiors. J Biol Chem 278 ,16528-33 (2003).

36. Weihoen, A., Lemberg, M.K., Ploegh, H.L.,Bogyo, M. & Maroglio, B. elease o signal pepideragmens ino he cyosol requires cleavage in heransmembrane region by a proease aciviy hais specifically blocked by a novel cyseine proeaseinhibior. J Biol Chem 275 , 30951-6 (2000).

37. Maroglio, B. & Golde, .E. Inramembrane-cleaving asparic proeases and disease: presenilins,

signal pepide pepidase and heir homologs. Hum Mol Gene 12 Spec No 2 , 201-6 (2003).

38. Friedmann, E. e al. SPPL2a and SPPL2bpromoe inramembrane proeolysis oNFalpha in acivaed dendriic cells o riggerIL-12 producion. Na Cell Biol 8 , 843-8 (2006).

39. Li, X. e al. Srucure o a presenilin amilyinramembrane asparae proease. Naure 493 ,56-61 (2013).

40. Fluhrer, . e al. e alpha-helical conen o heransmembrane domain o he Briish demeniaproein-2 (Bri2) deermines is processing bysignal pepide pepidase-like 2b (SPPL2b). J Biol Chem 287 , 5156-63 (2012).

41. Lemberg, M.K. & Maroglio, B. equiremens orsignal pepide pepidase-caalyzed inramembraneproeolysis. Mol Cell 10 , 735-44 (2002).

42. Lemberg, M.K. & Maroglio, B. On hemechanism o SPP-caalysed inramembraneproeolysis; conormaional conrol o pepide

bond hydrolysis in he plane o he membrane.FEBS Let 564 , 213-8 (2004).

43. McLauchlan, J., Lemberg, M.K., Hope, G. &Maroglio, B. Inramembrane proeolysis promoes

rafficking o hepaiis C virus core proein o lipiddroples. EMBO J 21 , 3980-8 (2002).

44. Loureiro, J. e al. Signal pepide pepidase isrequired or dislocaion rom he endoplasmicreiculum. Naure 441 , 894-7 (2006).

45. Schrul, B., Kapp, K., Sinning, I. & Dobbersein,B. Signal pepide pepidase (SPP) assembles

wih subsraes and misolded membraneproeins ino disinc oligomeric complexes. Biochem J 427 , 523-34 (2010).

46. Snyder, H.L. e al. wo novel roues oransporer associaed wih anigen processing(AP)-independen major hisocompaibiliycomplex class I anigen processing. J. Exp. Med. 186 , 1087-98 (1997).

47. Snyder, H.L., Bacik, I., Yewdell, J.W., Behrens,.W. & Bennink, J.. Promiscuous liberaion

o MHC-class I-binding pepides rom heC ermini o membrane and soluble proeinsin he secreory pahway. Eur. J. Immunol. 28 ,1339-46 (1998).

48. Puene, X.S., Sanchez, L.M., Overall, C.M. &Lopez-Oin, C. Human and mouse proeases: acomparaive genomic approach. Na Rev Gene 4 , 544-58 (2003).

49. Kirkin, V. e al. e Fas ligand inracellular domainis released by ADAM10 and SPPL2a cleavage in -cells. Cell Deah Differ 14 , 1678-87 (2007).

50. Beisner, D.. e al. e inramembrane

proease Sppl2a is required or B cell and DCdevelopmen and survival via cleavage o heinvarian chain. J Exp Med 210 , 23-30 (2013).

51. Bergmann, H. e al. B cell survival, surace BCand BAFF expression, CD74 meabolism, andCD8- dendriic cells require he inramembraneendopepidase SPPL2A . J Exp Med 210 , 31-40(2013).

52. Schneppenheim, J. e al. e inramembraneproease SPPL2a promoes B cell developmenand conrols endosomal raffic by cleavage o heinvarian chain. J Exp Med 210 , 41-58 (2013).

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SUPPLEME NTARY FIGUR ES AND TABLES

80.97%

7.63%

39.63%

Trh4 (anti-V5) Organelle staining Merge Colocalization

ER

Golgi

Mitochondria

T r h 4 ( a n t i - V 5 )

BIP

Giantin

Mitofilin

BIP

T r h 4

( a n t i - V 5 )

76.13%

ER

A

B

C

B 16.83%

A 97.08%

Golgi

ER

T r h 4 ( a n t i - V 5 )

BIP

Giantin

66,3%

ColocalizationOverlay

6,32%

ColocalizationOverlay

T r h 4 ( a n t i - V 5 )

BIP T r h 4 ( a n t i - V 5 )

ER Golgi

C

D

Giantin

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Supplementary Figure 2. Presentation of rh4-derived peptide is not affected by calpain 2silencing, but is SPP-dependent. (A) MA-S cells were incubaed wih he proease inhibior calpepina he indicaed concenraions or 1h. Cells were washed and incubaed wih a mild acid soluion odisrup MHC-I/pepide complexes rom he cell surace. Afer washing, cells were incubaed again wihcalpepin or addiional 6h o allow recovery o MHC-I/pepide complexes and hen incubaed overnigh

wih rh4-specific CL in he presence o Breelding A, o preven release o IFN-γ. Inracellular cyokinesaining was perormed and percenage o acivaed CL was deermined by flow cyomery. ConrolCL recognized he MuLV gag-leader pepide (CCLCLVFL) on MA cells. A he given calpepinconcenraions no oxiciy on arge cells was deeced. (B) Expression o he calpain amily member genes

was analysed in MA-S cells wih qPC. Expression levels were normalized o GAPDH housekeeping

gene and expressed relaive o he calpain 1 gene. Primer sequences are given in Suppl able I. (C)MA-S cells were ransduced wih leniviruses encoding shNA sequences ha arge calpain 2 or an

0 20 40 60 80 100 120

Stripping

60 µM

40 µM

20 µM

12 µM

CTL recognition (%)

Calpeptin

0.0

0.2

0.4

0.6

0.8

1.0

1.2

e m p t y v e c t o r

s h R N A c a l p a i n 2




s h R N A c a l p a i n 2

0

5

10

15

20

I F N γ


A B C

c a p n 1

c a p n 2

c a p n 3

c a p n 5

c a p n 6

c a p n 7

c a p n 8

c a p n 9

c a p n 1 0

c a p n 1 1

c a p n 1 2

c a p n 1 3

0

10

20

30

40

D

SPP SPPL2a SPPL2b SPPL2c SPPL30

500

1000

1500

2000

2500


number of targets number of targets

Trh4-CTL Control-CTL


0

20

40

60

Bone MarrowBrain

Liver

Lung

Lymph Node

Spleen

Thymus

E

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

0.0

0.3

0.6

0.9

1.2

1.5

1.8 empty vector

shRNA SPP

I F N γ


2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

1 2 0 0 0

0

4

8

12

16

F


s h R N A S P P

0.0

0.2

0.4

0.6

0.8

1.0

1.2

G

▶

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empy vecor, leading o decreased mNA levels o his gene. (D) CL recogniion o MA-S cells wihdownregulaed expression o calpain 2 was comparable o conrol MA-S arges. Daa are represenaiveo hree independen experimens. (E) Expression levels o SPP amily members in mouse organs. NArom complee mouse organs o wild ype C57BL/6 mice was exraced and expression o SPP amilymembers was deermined by qPC. Expression levels were normalized o GAPDH housekeeping geneand expressed relaive o he SPPL2c sample in liver. (F) Silencing o SPP in AP-deficien MCA cells

was reached by lenivirus-mediaed expression o shNA consrucs. Empy shNA vecor served asconrol. Efficiency o downregulaed levels o ranscrips was measured by qPC. (G) Cells wih silenced

SPP were esed or recogniion by rh4-specific CL or conrol CL reacive o an independen K b

-presened pepide. Daa are represenaive o hree independen experimens.

▶

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c o n t r o l

w t T r h 4

D e l # 1

E x t # 2

37

82

48

64

*

0

20

40

60

80

100

120

C T L r e c o g n i t i o n ( % )

# 1

# 6

# 3

# 5

# 2

# 7

# 8

# 9

# 1 0

# 4

w t

SDM construct

A

B

Supplementary Figure 3. Detection of V5-tagged rh4 constructs by western blot and CLrecognition efficiency of all side-directed mutagenesis constructs. (A) HeLa cells were ransienlyranseced wih V5-agged rh4 consrucs. e ag was inroduced a he N-erminus o he proein

(a aa posiion 33; ‘Del #1’ and ‘w rh4’) or as an exension a he C-erminus (‘Ex #2’), see Fig 5A.wo days laer he cells were lysed and proeins were separaed on SDS-page gel. Wesern blo analysis was perormed wih ani-V5 anibodies o confirm expression o hese consrucs afer ransecion.Daa are represenaive o hree independen experimens. Open riangle indicaes ull lengh rh4proein, closed riangle represens rh4 wihou he firs 32aa, prediced o be a leader sequence and* indicaes a non-specific band presen in all HeLa lysaes, serving as loading conrol. (B) e rh4gene was muaed a single posiions using sie-direced muagenesis. e consruc numbers reer ohe consrucs menioned in Figure 5C. ese rh4 consrucs were ransienly ranseced ino HeLacells ogeher wih he mouse MHC-I molecule D b. wo days laer HeLa cells were colleced andincubaed a differen arge concenraions wih rh4-specific CL. CL recogniion was deermined

by IFN-γ release in he supernaans and quanified by ELISA and he efficiency o CL recogniion wascalculaed as a percenage compared o he wild ype consruc using ‘area under he curve’ resuls o

original line graphs (see Figure 5D). Depiced daa are compiled rom hree independen experimensand means plus sandard error o he means are ploted.

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S u p p l e m e n t a r y a b l e 1 . P r i m e r s e q u

e n c e s f o r t h e q u a n t i t a t i v e P C R f o

r t h e m o u s e C a l p a i n f a m i l y .

N a

m e

G e n e I D

F o r w a r d p r i m e r

( 5 ’ - > 3 ’ )

R e v e r s e p r i m e r ( 5 ’ - >

3 ’ )

P r

o d . S

i z e

( b p )

C a l p a i n 1

1 2 3 3 3

T C T C C A T C T C C C G

C T C C T C C G

T C G A G

1 1 4

C a l p a i n 2

1 2 3 3 4

T C G G C A C A G A G G T

C G G A G G T A A

G A A G G A C

1 1 6

C a l p a i n 3

1 2 3 3 5

G A G G A C C G A G G A A C G

G C G A A G C C A A A G G A A G

1 0 1

C a l p a i n 5

1 2 3 3 7

C A C G A C G A G G C A A G A G

C T G A G G A G G

C A C G A

8 5

C a l p a i n 6

1 2 3 3 8

G G A A C G A C C T G G A C A

T C A T G G C A A C T C T C C

1 2 1

C a l p a i n 7

1 2 3 3 9

A C C G G G G C G G C C G

C C A T G C C

C C C G G C C A T

9 0

C a l p a i n 8

1 7 0 7 2 5

A C A C A C C A C C A G A A G G

C C A G C A C A G A G A G C

1 3 1

C a l p a i n 9

7 3 6 4 7

G G A C G G A C A C C G A G A G

C G G A A C G C C G A G A C

9 7

C a l p a i n 1 0

2 3 8 3 0

A G G G A C T C A T C C T A G G

C G C C G C C A G G A A

1 2 1

C a l p a i n 1 1

2 6 8 9 5 8

G G A G G G A A C A G C C A C

A A G C A G G A A G

C A G C A C

9 1

C a l p a i n 1 2

6 0 5 9 4

G C A A C C A C A C T C C A

C T C A C A C G

C A C C

1 4 3

C a l p a i n 1 3

3 8 1 1 2 2

G C A C T G G C C C A T C C

G A T G C G G C T C A C G

1 3 6

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Alternative Antigen Processing and Presentation Pathways by Tumors - 03

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Transcript of Alternative Antigen Processing and Presentation Pathways by Tumors - 03